Prospective identification and characterization of breast cancer stem cells

ABSTRACT

Human breast tumors contain hetrogeneous cancer cells. using an animal xenograft model in which human breast cancer cells were grown in immunocompromised mice we found that only a small minority of breast cancer cells had capacity to form new tumors. The ability to form new tumors was not a slochastic property, rather certain populations of cancer cells were depleted for the ability to form new tumors, while other populations were enriched for the ability to form new tumors. Tumorigenic cells could be distinguished from non-tumorigenic cancer cells based on surface marker expression. We prospectively identified and isolated the tumorigenic cells as CD44 30 CD24 −/low LINEAGE A few as 100 cells from this population were able to form tumors the animal xenograft model, while tens of thousands of cells from non-tumorigenic populations failed to form tumors. The tumorigenic cells could be serially passaged, each time generating new tumors containing and expanded numbers of CD44 +CD24  Lineage tumorigenic cells as well as phenotypically mixed populations of non-tumorigenic cancer cells. This is reminiscent of the ability of normal stem cells to self-renew and differentiate. The expression of potential therapeutic targets also differed between the tumorigenic and non-tumorigenic populations. Notch activation promoted the survival of the tumorigenic cells, and a blocking antibody against Notch 4 induced tumorigenic breast cancer cells to undergo apoptosis.

TECHNICAL FIELD OF THE INVENTION

This invention relates general to the investigation or analysis ofbiological materials by determining their chemical or physicalproperties, and in particular to the diagnosis and treatment of cancer.

BACKGROUND ART

Breast cancer is the most common cancer in women, but metastatic breastcancer is still incurable. Despite advances in detection and treatmentof metastatic breast cancer, mortality from this disease remains highbecause current therapies are limited by the emergence oftherapy-resistant cancer cells. As a result, metastatic breast cancerremains an incurable disease using current treatment strategies.

In solid tumors generally, only a small proportion of the tumor cellsare able to form colonies in an in vitro clonogenic assay. Large numbersof cells must typically be transplanted to form tumors in vivo. Theseobservations have been explained by a stochastic model in which eachtumor cell has the capacity to proliferate and form new tumors but onlya small proportion of the cells is able to exhibit this capacity at anyone time.

Alternatively, only a rare subset of solid tumor cells may have thecapacity to significantly proliferate or form new tumors, but cellswithin this subset may do so very efficiently. If only a small,identifiable subset of solid tumors cells possesses the capacity toproliferate and form new solid tumors, this would have importantimplications for cancer therapy. To eradicate solid tumors, it would benecessary to kill this subpopulation of cells.

The prospective identification and isolation of hematopoietic stem cellsand nervous system stem cells has brought about rapid advances in ourunderstanding of these cells. Thus, if it is possible to prospectivelyidentify and isolate a tumorigenic cell population, it would then bepossible to much more effectively focus the development anti-solid tumortherapeutics and diagnostics.

DISCLOSURE OF THE INVENTION

The invention is based upon the discovery that a small percentage oftumorigenic cells within an established solid tumor have the propertiesof stem cells. These solid tumor stem cells give rise both to more solidtumor stem cells and to the majority of cells in the tumor, cancer cellsthat have lost the capacity for extensive proliferation and the abilityto give rise to new tumors. Thus, solid tumor cell heterogeneityreflects the presence of a variety of tumor cell types that arise from asolid tumor stem cell.

This invention provides a way that anti-cancer therapies can bedirected, both generally and now specifically directed, against thesolid tumor stem cells. The previous failure of cancer therapies tosignificantly improve outcome has been due in part to the failure ofthese therapies to target the solid tumor stem cells within a solidtumor that have the capacity for extensive proliferation and the abilityto give rise to all other solid tumor cell types. Effective treatment ofsolid tumors thus requires therapeutic strategies that are able totarget and eliminate the tumorigenic subset of solid tumor cells, i.e.,the solid tumor stem cells, by the direct targeting of therapeutics tothe solid tumor stem cells. Accordingly, the invention provides a methodfor reducing the size of a solid tumor, by contacting the cells of thesolid tumor with a therapeutically effective amount of an agent directedagainst a Notch4 polypeptide. Inhibition of Notch4-signaling impairs thegrowth of the solid tumor stem cells. The invention also provides amethod for reducing the size of a solid tumor; by contacting the cellsof the solid tumor with a therapeutically effective amount of an agentthat modulates the activity of Maniac Fringe.

The invention provides in vivo and in vitro assays of solid tumor stemcell function and cell function by the various populations of cellsisolated from a solid tumor. The invention provides methods for usingthe various populations of cells isolated from a solid tumor (such as apopulation of cells enriched for solid tumor stem cells) to identifyfactors influencing solid tumor stem cell proliferation. By the methodsof the invention, one can characterize the phenotypically heterogeneouspopulations of cells within a solid tumor. In particular, one canidentify, isolate, and characterize a phenotypically distinct cellpopulation within a tumor having the stem cell properties of extensiveproliferation and the ability to give rise to all other tumor celltypes. Solid tumor stem cells are the truly tumorigenic cells that arecapable of re-establishing a tumor following treatment.

The invention thus provides a method for selectively targetingdiagnostic or therapeutic agents to solid tumor stem cells. Theinvention also provides an agent, such as a biomolecule, that isselectively targeted to solid tumor stem cells.

In its several aspects, the invention usefully provides methods forscreening for anti-cancer agents; for the testing of anti-cancertherapies; for the development of drugs targeting novel pathways; forthe identification of new anti-cancer therapeutic targets; theidentification and diagnosis of malignant cells in pathology specimens;for the testing and assaying of solid tumor stem cell drug sensitivity;for the measurement of specific factors that predict drug sensitivity;and for the screening of patients (e.g., as an adjunct for mammography).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the isolation of tumorigenic cells. Flow cytometry was usedto isolate subpopulations of Tumor 1 (T1; FIG. 1 a, FIG. 1 b), Tumor 3(T2; FIG. 1 c), Tumor 5 (T5; FIG. 1 d), Tumor 6 (T6; FIG. 1 e) and Tumor7 (T7; FIG. 1 f) cells, which were tested for tumorigenicity in NOD/SCIDmice. T1(FIG. 1 b) and T3 (FIG. 1 c) had been passaged (P) once inNOD/SCID mice. The rest of the cells were frozen or unfrozen samplesobtained directly after removal from a patient (UP). Cells were stainedwith antibodies against CD44, CD24, LINEAGE markers, and mouse-H2K (forpassaged tumors obtained from mice), and 7AAD. Dead cells (7AAD⁺), mousecells (H2K⁺) and LINEAGE⁺ normal cells were eliminated from allanalyses. Each plot depicts the CD24 and CD44 staining patterns of livehuman LINEAGE⁻ cancer cells, and the frequency of the boxed tumorigeniccancer population as a percentage of cancer cells/all cells in eachspecimen is shown. Tumor 3 (T3) cells were stained with Papanicolaoustain and examined microscopically (100× objective). Both thenon-tumorigenic (FIG. 1 g) and tumorigenic (FIG. 1 h) populationscontained cells with a neoplastic appearance, with large nuclei andprominent nucleoli. Histology from the CD24⁺ injection site (FIG. 1 i;20× objective magnification) revealed only normal mouse tissue while theCD24^(−/low) injection site (FIG. 1 j; 40× objective magnification)contained malignant cells (FIG. 1 k). A representative tumor in a mouseat the CD44⁺CD24^(−/low)LINEAGE³¹ injection site, but not at theCD44⁺CD24⁺ LINEAGE⁻ injection site.

Supplemental FIG. 1 shows the expression of Notch4 by MCF-7 and MCF-10cells. MCF-7 cells (Supplemental FIG. 1 a) and MCF-10 cells(Supplemental FIG. 1 b) were stained with the anti-Notch4 antibody. T1cells and MCF-7 cells express higher levels of the protein than MCF-10cells. (Supplemental FIG. 1 c) RT-PCR was done using nested primers todetect expression of Notch4 MRNA. Notch4 was expressed by MCF-7 cells,and MCF-10 cells. The message was not detected when reversetranscriptase (RT) was omitted from the reaction (MCF10/no RT). Weconfirmed that the MCF-7 cells expressed Notch4 at both the RNA andprotein levels. These data were independently confirmed using twodifferent pairs of intron spanning Notch4-specific PCR primers. Note, itis possible that different sublines of “NCF-7” cells in circulation candiffer in their expression of Notch4. Osbome CK et al., Breast CancerResearch & Treatment. 9: 111-121 (1987).

FIG. 2 shows the phenotypic diversity in tumors arising from solid tumorstem cells. The plots depict the CD24 and CD44 or ESA staining patternsof live human LINEAGE⁻ cancer cells from Tumor 1 (T1; FIG. 2 a, FIG. 2 cand FIG. 2 e) or Tumor 2 (T2; FIG; 2 b, FIG. 2 d and FIG. 2 f). T1CD44⁺LINEAGE⁻ cells (FIG. 2 a) or T2 LINEAGE⁻ cells (FIG. 2 b) wereobtained from tumors that had been passaged once in NOD/SCID mice.ESA⁺CD44⁺CD24^(−/low) LINEAGE⁻ tumorigenic cells from T1(FIG. 2 c) orCD44⁺CD24^(−/low) LINEAGE⁻ tumorigenic cells from T2 (FIG. 2 d) wereisolated and injected into the breasts of NOD/SCID mice. Plots shown inFIG. 2 e and FIG. 2 f depict analyses of the tumors that arose fromthese cells. In both cases, the tumorigenic cells formed tumors thatcontained phenotypically diverse cells similar to those observed in theoriginal tumor. The cell cycle status of the ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻ tumorigenic cells (FIG. 2 g) and the remaining LINEAGE⁻non-tumorigenic cancer cells (FIG. 2 h) isolated from T1were determinedby hoechst 33342 staining of DNA content, according to the method ofMorrison SJ & Weissman IL, Immunity 1: 661-673 (1994). The tumorigenicand non-tumorigenic cell populations exhibited similar cell cycledistributions.

FIG. 3 shows that blocking antibodies against Notch4 inhibited colonyformation by solid tumor stem cells. FIG. 3 a shows Notch4 expression byT1tumorigenic breast cancer cells. Tumorigenic (CD44⁺CD24^(−/low)LINEAGE³¹ ) T1cells that had been passaged once in NOD/SCID mice werestained with the anti-Notch4 antibody. FIG. 3 b shows colonyformation/unsorted 20,000 T1 cells grown for 14 days in the indicatedtissue culture medium supplemented with Fc antibody (control);polyclonal anti-Notch4 antibody (Ab); polyclonal anti-Notch4 antibodyplus blocking peptide (Ab+Block); Delta-Fc (Delta); Delta plusanti-Notch4 Ab (Delta+Ab); and Delta plus polyclonal anti-Notch4antibody plus blocking peptide (Delta+Ab+B). Soluble Delta-Fc (Delta)stimulated colony formation (p<0.001), while the polyclonal anti-Notch4antibody (Ab) inhibited colony formation in the presence of Delta-Fc(Delta+Ab) (p<0.001). When the antibody was pre-incubated with thepeptide used to generate the anti-Notch4 antibody, the inhibitory effectof the antibody was nearly completely reversed (Ab+Block;Delta+Ab+Block; p<0.001). FIG. 3 b is a Notch pathway reporter geneassay showing that soluble delta-Fc (Delta) activated Notch relative tocontrol Fc construct (Control). Anti-Notch4 polyclonal antibody (Ab)inhibited Notch activation, even in the presence soluble Delta-Fc(Delta+Ab). Addition of a blocking peptide against which the polyclonalantibody was made (Block) partially reversed the ability of the antibodyto inhibit Notch activation (Delta+Ab+Block). In FIG. 3 d,ESA⁺CD44⁺CD24^(−/low) LINEAGE⁻ tumorigenic cells were isolated fromfirst or second passage T1tumor. The indicated number of cells wereinjected into the area of the mammary fat pads of mice in control bufferor after being. incubated with a polyclonal anti-Notch4 antibody. Tumorformation was monitored over a five-month period. Note that tumorformation by 500 antibody-treated cells was delayed by an average ofthree weeks.

FIG. 4 shows that Notch4 signaling provides a survival signal totumor-initiating cells. FIG. 4 a shows the cell cycle status of controlMCF-7 cells (shaded) and MCF-7 cells 24 hrs after exposure to theanti-Notch4 antibody (open) was determined by PI staining of DNA contentaccording to the methods of Clarke M F et al., Proc. Natl. Acad. Sci.USA 92: 11024-11028 (1995) and Ryan J J et al., Mol. & Cell. Biol. 1:711-719 (1993). Each group exhibited similar cell cycle distributions.FIG. 4 b shows PI³⁰ apoptotic MCF-10, MCF-7,ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻ tumorigenic Tumor 1 (T1) cells grown inmedia for 48 hours, or H2K⁻ Tumor 7 (T7), Tumor 8 (T8), or Tumor 10(T10) cells grown in media for 5 days with (+Ab) or without theanti-Notch4 antibody were identified by flow cytometry. The timing ofthe onset of apoptosis after antibody addition was similar to that seenafter some other death signals. Clarke M F et al., Proc. Natl. Acad.Sci. USA 92: 11024-11028 (1995)(bcl-xs); Ryan J J et al., MoL & Cell.BioL 1: 711-719 (1993) (p53)). Note that the antibody was lethal to theT1and T10 cells. FIG. 4 c shows that at forty-eight hours after exposureto the anti-Notch4 antibody, the percentage of cells expressingactivated caspase 3 and/or 7, as measured by flow cytometry using thefluorogenic substrate CaspoTag™, was markedly increased inT1tumor-initiating cells and MCF-7 cells, but not MCF-10 cells, ascompared to control cells. Tumor 1 (T1) tumorigenic(ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻) cells were isolated by flow cytometry asdescribed in TABLE 3.

MODES FOR CARRYING OUT THE INVENTION

Introduction. By this invention, the principles of normal stem cellbiology have been applied to isolate and characterize solid tumor stemcells generally. Solid tumor stem cells are defined structurally andfunctionally as described herein; using the methods and assays similarto those described below. Solid tumor stem cells undergo “self-renewal”and “differentiation” in a chaotic development to form a tumor, giverise to abnormal cell types, and may change over time as additionalmutations occur. The functional features of a solid tumor stem cell arethat they are tumorigenic, they give rise to additional tumorigeniccells (“self-renew”), and they can give rise to non-tumorigenic tumorcells (“differentiation”). The developmental origin of solid tumor stemcells can vary between different types of solid tumor cancers.Typically, solid tumors are visualized and initially identifiedaccording to their locations, not by their developmental origin.Accordingly, one can use the method of the invention, employing themarkers disclosed herein, which are consistently useful in the isolationor identification of solid tumor stem cells in a majority of patients.

Examples of solid tumors from which solid tumor stem cells can beisolated or enriched for according to the invention include sarcomas andcarcinomas such as, but not limited to: fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. The invention is particularly applicable to sarcomas andepithelial cancers, such as ovarian cancers and breast cancers.

“Enriched”, as in an enriched population of cells, can be defined basedupon the increased number of cells having a particular marker in afractionated set of cells as compared with the number of cells havingthe marker in the unfractionated set of cells. However, the term“enriched” can be preferably defined by tumorigenic function as theminimum number of cells that form tumors at limit dilution frequency intest mice. The solid tumor stem cell model provides the linkage betweenthese two definitions of (phenotypic and functional) enrichment.

In particular, we have found that breast cancers contain heterogeneouspopulations of neoplastic cells. Using a xenograft model in which humanbreast cancer cells were grown in immunocompromised mice, we found thatonly a small minority of breast cancer cells had the capacity to formnew tumors. The ability to form new tumors was not a stochasticproperty. Rather, certain populations of cancer cells were depleted forthe ability to form new tumors while other populations were enriched forthe ability to form new tumors. Indeed, we could consistently predictwhich cells would be most tumorigenic based on surface markerexpression.

Using the methods of the invention, we prospectively identified andisolated the tumorigenic cells as CD44⁺CD24^(−/low)LINEAGE⁻. As few as100 cells from this population were able to form tumors inimmunocompromised mice, while tens of thousands of cells fromnon-tumorigenic populations failed to form tumors. TheCD44⁺CD24^(−/low)LINEAGE⁻ cells displayed stem cell-like properties inthat they were capable of generating new tumors containing additionalCD44⁺CD24^(−/lo)LINEAGE⁻ tumorigenic cells as well as the phenotypicallymixed populations of non-tumorigenic cells present in the originaltumor. The expression of potential therapeutic targets also differedbetween the tumorigenic and non-tumorigenic populations of cancer.

Inhibition of Notch4-signaling impaired the growth of the breast cancerstem cells in vitro and in vivo. Effective treatment of solid tumorsthus requires therapeutic strategies that are able to target andeliminate the tumorigenic subset of solid tumor cells, i.e., the solidtumor stem cells, by the direct targeting of therapeutics to the solidtumor stem cells.

Animal xenograft model. To test whether solid cancer cells vary in theirpotential to form new tumors according to the predictions of cancer cellheterogeneity models, we developed an animal xenograft model in whichprimary or metastatic human breast cancers could efficiently andreproducibly be grown and analyzed in immunodeficient mice. We used amodification of the NOD/SCID immunodeficient mouse model, in which humanbreast cancers were efficiently propagated in the area of the mousemammary fat pad. See, Sakakibara T et al., Cancer J. Sci. Am. 2: 291-300(1996). See also, published PCT patent application WO 02/12447, theentire contents of which are incorporated herein by reference.

Thus, the invention provides an animal xenograft model in which toestablish tumors by the injection of solid tumor cells into a hostanimal. The host animal can be a model organism such as nematode, fruitfly, zebrafish; preferably a laboratory mammal such as a mouse (nudemouse, SCID mouse, NOD/SCID mouse, Beige/SCID Mouse), rat, rabbit, orprimate. The severely immunodeficient NOD-SCID mice were chosen asrecipients to maximize the participation of injected cells.Immunodeficient mice do not reject human tissues, and SCID and NOD-SCIDmice have been used as hosts for in vivo studies of human hematopoiesisand tissue engraftinent. McCune et al., Science 241: 1632-9 (1988);Kamel-Reid & Dick, Science 242: 1706-9 (1988); Larochelle et al., Nat.Med. 2: 1329-37 (1996). In addition, Beige/SCID mice also have beenused. NOD/SCID mice have previously been validated as in vivo models forthe growth of normal human hematopoietic stem cells (Larochelle A etal., Nature Medicine 2: 1329-1337 (1996); Peled A et al., Science 283:845-8 (1999); Lapidot T et al., Science 255: 1137-1141 (1992)) humanneural stem cells (Uchida N et al., Proc. Natl. Acad. Sci. USA97:14720-5 (2000)) and human acute myelogenous leukemia (AML) stem cells(Lapidot T et al., Nature 17: 367:645-648. (1994); Bonnet D & Dick J E,Nature Medicine 3: 730-737 (1997)). Another useful mouse is the β2microglobin deficient NOD/SCID mouse. Kollet O et al., Blood 95:3102-3105 (2000).

Some previous clonogenic in vitro assays of cancer cells were difficultto interpret since cells from different tumors proliferated to differentextents and only occasionally yielded cells that could be seriallypassaged indefinitely (immortal cells). Similarly, some previous in vivoassays of tumorigenicity were difficult to interpret because cancercells from some patients engrafted while pathologically similar cancercells from other patients failed to engraft. By contrast, the animalxenograft model of this invention permitted tumor formation by all thepatient samples that were tested.

In the assays described below, 8-week old female NOD-SCID mice wereanesthetized and then injected IP with etoposide (30 mg/kg). At the sametime, estrogen pellets were placed subcutaneously on the back of theneck using a trocar. All tumor injections/implants were performed fivedays after this procedure. For the implantation of fresh specimens,samples of human breast tumors were received within an hour after thesurgeries. The tumors were minced to yield 2-mm³ pieces. A 2-mm incisionwas then made in the mouse and a 2-mm piece of a primary tumor wasinserted or 10⁷ pleural effusion cells were injected into the breast. A6-0 suture was wrapped twice around the breast and nipple to hold theimplanted pieces in place. Nexaban was used to seal the incision andmice were monitored weekly for tumor growth. For the injection of cancercells from pleural effusions, cells were received shortly afterthoracentesis and washed with HBSS. Viable cell numbers were countedduring sorting and verified using a hemocytometer. After centrifugation,the indicated number of cells were suspended in 100 μl of a serumfree-RPMI/Matrigel® mixture (1:1 volume). A nick was made approximately1-cm form the nipple, and an 18-gauge needle was inserted and tunneledinto the subcutaneous tissue immediately under the nipple. The cellswere then injected in the area of the mammary fat pad. The site of theneedle injection was sealed with Nexaban to prevent cell leakage.

Other general techniques for formulation and injection of cells into theanimal xenograft model may be found in Remington's PharmaceuticalSciences, 20th ed. (Mack Publishing Co., Easton, Pa.). Suitable routesmay include parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections, just to name a few. For injection, the agents ofthe invention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. For such transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

In the assays discussed below, the animals were injected with unsortedT1 and T3 cells, and a 2-mm piece of T2. Injected ells from T4-T9 wereisolated by flow cytometry as described in FIG. 1 and TABLE 3. Solidtumor cells for injection were obtained from a primary breast tumor (T2)as well as from metastatic pleural effusions (T1, T3-T9). Some assayswere performed on cells after they had been passaged once in mice(Passage 1; see, TABLE 3 below) while other assays were performed onunpassaged fresh or frozen tumor samples obtained directly from patients(Unpassaged; see, TABLE 3 below). For cell culture, Passage-1 primarybreast cancer cells were plated in triplicate 12-well dishes in HAM-F12medium supplemented with Fetal Bovine Serum (1%), Insulin (5 μg/ml),Hydrocortisone (1 μg/ml), EGF (10 μg/ml), Choleratoxin (0.1 μg/ml),Transfenrin and Selenium (GIBCO BRL, recommended dilutions), pen/strep,and fungizone (Gibco/BRL). Culture medium was replaced once every twodays.

As shown in TABLE 1 below, all of the solid tumor specimens that wereavailable to us engrafted in the animal xenograft model. Breast cancercells were obtained from nine different patients (designated tumors 1-9;T1-T9) and grown in the animal xenograft model model. TABLE 1Engraftment of Solid Tumor Cells into the Animal Xenograft Model TumorTumor formation Tumor origin in mice Serial passage in mice T1Metastasis Yes Yes T2 Breast primary Yes Yes T3 Metastasis Yes Yes T4Metastasis Yes No T5 Metastasis Yes Yes T6 Metastasis Yes Yes T7Metastasis Yes Yes T8 Metastasis Yes Yes T9 Metastasis Yes Yes

The tumors passaged in the animals contained heterogeneous cancer cellsthat were phenotypically similar to the cancer cells present in theoriginal tumors from patients (see, e.g., FIG. 1 a and FIG. 1 b),including both tumorigenic and non-tumorigenic fractions. This resultdemonstrates that the environment of the animal xenograft model was notincompatible with the survival of the non-tumorigenic cell fractions.Both the tumorigenic and non-tumorigenic fractions of cancer cellsexhibited a similar cell-cycle distribution in mouse tumors (FIG. 2 gand FIG. 2 h), demonstrating that the non-tumorigenic cells were able todivide in mice.

In summary, we did not encounter a specimen from which a significantnumber of cancer cells could be recovered that then failed to engraft.Only one sample failed to serially passage in the mice. Thus, the tumorsand tumorigenic cells characterized here are representative of all thebreast cancer specimens that were available to us, rather than a subsetthat was selected for the ability to grow in the assay. Moreover, wehave used the animal xenograft model to grow sarcoma cells. Thus, theanimal xenograft model reliably supports the engraftment of clonogenichuman progenitors, i.e., solid tumor stem cells.

Characterization of tumorigenic solid tumor stem cells. As describedabove, solid tumor stem cells can be operationally characterized by cellsurface markers. These cell surface markers can be recognized byreagents that specifically bind to the cell surface markers. Forexample, proteins, carbohydrates, or lipids on the surfaces of solidtumor stem cells can be immunologically recognized by antibodiesspecific for the particular protein or carbohydrate (for constructionand use of antibodies to markers, see, Harlow, Using Antibodies: ALaboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,1999)). The set of markers present on the cell surfaces of solid tumorstem cells (the “cancer stem cells” of the invention) and absent fromthe cell surfaces of these cells is characteristic for solid tumor stemcells. Therefore, solid tumor stem cells can be selected by positive andnegative selection of cell surface markers. A reagent that binds to asolid tumor stem cell is a “positive marker” (i.e., a marker present onthe cell surfaces of solid tumor stem cells) that can be used for thepositive selection of solid tumor stem cells. A reagent that binds to asolid tumor stem cell “negative marker” (i.e., a marker not present onthe cell surfaces of solid tumor stem cells but present on the surfacesof other cells obtained from solid tumors) can be used for theelimination of those solid tumor cells in the population that are notsolid tumor stem cells (i.e., for the elimination of cells that are notsolid tumor stem cells).

The discrimination between cells can be based upon the detectedexpression of cell surface markers is by comparing the detectedexpression of the cell surface marker as compared with the meanexpression by a control population of cells. For example, the expressionof a marker on a solid tumor stem cell can be compared to the meanexpression of the marker by the other cells derived from the same tumoras the solid tumor stem cell. Other methods of discriminating amongcells by marker expression include methods of gating cells by flowcytometry based upon marker expression (see, Givan A, Flow Cytometry:First Principles, (Wiley-Liss, New York, 1992); Owens M A & Loken M R,Flow Cytometry: Principles for Clinical Laboratory Practice,(Wiley-Liss, New York, 1995)).

A “combination of reagents” is at least two reagents that bind to cellsurface markers either present (positive marker) or not present(negative marker) on the surfaces of solid tumor stem cells, or to acombination of positive and negative markers. The use of a combinationof antibodies specific for solid tumor stem cell surface markers resultsin the method of the invention being useful for the isolation orenrichment of solid tumor stem cells from a variety of solid tumors,including sarcomas, ovarian cancers, and breast tumors. Guidance to theuse of a combination of reagents can be found in published PCT patentapplication WO 01/052143, incorporated by reference.

To prepare cells for flow cytometric analysis in the assays describedherein, single cell suspensions of solid tumors or pleural effusionswere made by mincing solid tumors and digesting them with 200 μ/ml ofcollagenase 3 (Worthington) in M119 medium (Gibco/BRL, Rockville, Md.USA) for 2-4 hours at 37° C. with constant agitation. Antibodiesincluded anti-CD44, anti-CD24, anti-B38.1, anti-EGFR, anti-HER2/neu,anti-ESA-FITC (Biomeda, Calif. USA), anti-H2K, and goat-anti-humanNotch4 (Santa Cruz Products, Santa Cruz, Calif. USA). CD44 (Saddik M &Lai R, J. Clin. Pathol. 52(11): 862-4 (1999)) and CD24 (Aigner S et al.,Blood: 89(9): 3385-95 (1997)) are adhesion molecules. B38.1 has beendescribed as a breast/ovarian cancer-specific marker (Ahrens T et al.,Oncogene 20: 3399-408, (2001); Uchida N et al., Proc. Natl. Acad. Sci.USA 97: 14720-5 (2000); Kufe D W et al., Cancer Research 43: 851-857(1983)). LINEAGE marker antibodies were anti-CD2, -CD3-CD10, -CD16,-CD18, -CD31, -CD64 and -CD140b. Unless noted, antibodies are availablefrom Pharmingen (San Diego, Calif. USA). Antibodies were directlyconjugated to various fluorochromes depending on the assay. Dissociatedtumor cells were stained with anti-CD44, anti-CD24, anti-B38.1,anti-EGFR, anti-HER2/neu, anti-ESA, anti-H2K, Streptavidin-Phar-red,goat-anti-human Notch4, donkey anti-goat Ig-FITC,anti-LINEAGE-Cytochrome (LINEAGE antibodies were anti-CD2, -CD3-CD10,-CD14, -CD18, -CD31, -CD64 and -CD140b) each directly conjugated to afluor except H2k which was biotinylated. Mouse cells and/or LINEAGE⁺cells can be eliminated by discarding H2K⁺ (class I MHC) cells orLINEAGE⁺ cells during flow cytometry. Dead cells can be eliminated usingthe viability dye 7-AAD. Flow cytometry and cell sorting can beperformed on a FACSVantage (Becton Dickinson, San Jose, Calif. USA).Data files can be analyzed using Cell Quest software (Becton Dickinson).

We found that breast cancer cells were heterogeneous with respect toexpression of a variety of cell surface-markers including CD44, CD24,and B38.1.

To determine whether these markers could distinguish tumorigenic fromnon-tumorigenic cells, flow cytometry was used to isolate cells thatwere positive or negative for each marker from first passage T1 or T2cells. Cells were isolated by flow cytometry as described in FIG. 1,based upon expression of the indicated marker and assayed for theability to form tumors after injection into the mammary fat pads ofNOD/SCID mice. For twelve weeks, mice were examined weekly for tumors byobservation and palpation. Then, all mice were necropsied to look forgrowths at injection sites that were too small to palpate. A “palpabletumor” is known to those in the medical arts as a tumor that is capableof being handled, touched, or felt. All tumors were readily apparent byvisual inspection and palpation except for tumors from the CD24⁺population which were only detected upon necropsy.

When 200,000-800,000 cells of each population were injected, allinjections of CD44⁺ cells (8/8), B38.1³⁰ cells (8/8), or CD24^(−/low)cells (12/12) gave rise to visible tumors within twelve weeks ofinjection, but none of the CD44⁻ cell (0/8), or B38.1⁻ cell (0/8)injections formed detectable tumors (TABLE 2). The ratio of the numberof tumors that formed/the number of injections that were performed isindicated for each population. TABLE 2 Tumorigenicity of DifferentPopulations of Solid Tumor Stem Cells # tumors/# of injectionsCells/injection 8 × 10⁵ 5 × 10⁵ 2 × 10⁵ T1 cells CD44⁻ 0/2 0/2 — CD44⁺2/2 2/2 — B38.1⁻ 0/2 0/2 — B38.1⁺ 2/2 2/2 — CD24⁺ — — 1/6 CD24⁻ — — 6/6T2 cells CD44⁻ 0/2 0/2 — CD44⁺ 2/2 2/2 — B38.1⁻ 0/2 0/2 — B38.1⁺ 2/2 2/2— CD24⁺ — — 1/6 CD24⁻ — — 6/6

Although no tumors could be detected by palpation in the locationsinjected with CD24⁺ cells, two of twelve mice injected with CD24⁺ cellsdid contain small growths at the injection site that were detected uponnecropsy. These growths most likely arose from the 1-3% of CD24⁻ cellsthat invariably contaminated the sorted CD24⁺ cells, or alternativelyfrom CD24⁺ cells with reduced proliferative capacity (TABLE 2). Becausethe CD44⁺ cells were exclusively B38.1⁺, we focused on the CD44 and CD24markers in subsequent assays.

Several antigens associated with normal cell types (LINEAGE markers CD2,CD3, CD10, CD16, CD18, CD31, CD64, and CD140b) were found not to beexpressed by the cancer cells based on analyses of tumors that had beenpassaged multiple times in mice. By eliminating LINEAGE⁺ cells fromunpassaged or early passage tumor cells, normal human leukocytes,endothelial cells, mesothelial cells and fibroblasts were eliminated. Bymicroscopic examination, the LINEAGE⁻ tumor cells consistently had theappearance of neoplastic cells (FIG. 1 g and FIG. 1 h).

An average of 15% (range from 8% to 26%) of the LINEAGE⁻ cancer cells intumors or pleural effusions were CD44⁺CD24^(−/low) (FIG. 1 ato FIG. 1f). CD44⁺CD24^(−/low)LINEAGE⁻ cells or other populations of LINEAGE⁻cancer cells that had been isolated from nine patients were injectedinto the breasts of mice (TABLE 3). When injecting unfractionatedpassaged T1or T2 cells, 50,000 cells consistently gave rise to tumors,but 10,000 cells gave rise to tumors in only a minority of cases (TABLE3). In contrast, as few as 1,000 T1 or T2 CD44³⁰ CD24^(−/low)LINEAGE⁻cells gave rise to tumors in all cases (TABLE 3). For T1 and T2, up to20,000 cells that were CD44⁺ LINEAGE⁻ but CD24⁺ failed to form tumors(FIG. 1 k). These data suggest that the CD44⁺CD24^(−/low) LINEAGE⁻population is 10-50 fold enriched for the ability to form tumors inNOD/SCID mice relative to unfractionated tumor cells.

Whether the CD44⁺CD24^(−/low) LINEAGE⁻ cells were isolated from passagedtumors (T1, T2, T3) or from unpassaged tumors (T1, T4-T6, T8, T9), thecells were enriched for tumorigenic activity (TABLE 3). Note that T7 wasthe only one of nine cancers that we tested that did not fit thispattern (TABLE 3; see, below). CD44⁺CD24^(−/low)LINEAGE⁻ andCD24⁺LINEAGE⁻ cancer cells were consistently depleted of tumorigenicactivity in both passaged and unpassaged samples (TABLE 3). Therefore,the xenograft and unpassaged patient tumors were composed of similarpopulations of phenotypically diverse cell types, and in both cases onlythe CD44+CD24 ^(−/low)LINEAGE⁻ cells had the capacity to proliferate toform new tumors (p<0.001).

TABLE 3 shows that tumorigenic breast cancer cells were highly enrichedin the ESA⁺CD44⁺CD24^(−/low) population. Cells were isolated from firstpassage (designated Passage 1) Tumor 1, Tumor 2 and Tumor 3, secondpassage Tumor 3 (designated Passage 2), unpassaged T1, T4, T5, T6, T8and T9 (designated Unpassaged), or unpassaged T7 cells (designatedunpassaged T7). The indicated number of cells of each phenotype wasinjected into the breast of NOD/SCID mice. TABLE 3 Tumorigenicity ofDifferent Populations of Solid Tumor Stem Cells 500,000 100,000 50,00020,000 10,000 5,000 1,000 500 200 100 Passage 1 Unsorted 8/8 8/8 10/10 3/12  0/12 CD44⁺CD24⁺  0/10  0/10  0/14  0/10 CD44⁺CD24^(−/low) 10/1010/10 14/14 10/10 CD44⁺CD24^(−/low)ESA⁺  10/10* 4/4 4/4 1/6CD44⁺CD24^(−/low)ESA⁻  0/10* 0/4 0/4 0/6 Passage 2 CD44⁺CD24⁺ 0/9CD44⁺CD24^(−/low) 9/9 Unpassaged CD44⁺CD24⁺ 0/3 0/4 0/8  1/13 0/2CD44⁺CD24^(−/low) 3/3 4/4 11/13 1/1 CD44⁺CD24^(−/low)ESA⁺ 2/2 2/2CD44⁺CD24^(−/low)ESA⁻  2/2^(#) 0/2 Unpassaged T7 CD44⁺CD24^(−/low) 2/2CD44⁺CD24⁺ 2/2 CD44⁻CD24⁺ 0/2 MCF-7 cells 10/10 10/10  0/20#Tumor formation by T5 ESA⁻CD44⁺CD24^(−/low)LINEAGE⁻ cells was delayedby 2-4 weeks.*2,000 cells were injected in these experiments. In addition to themarkers that are shown, all sorted cells in all experiments wereLINEAGE⁻, and the tumorigenic cells from T1, T2, and T3 were furtherselected as B38.1⁺.

The frequency of tumorigenic cells calculated by the modified maximumlikelihood analysis method is ˜5/10⁵, according to the methods of PorterE H & Berry R J, Br. J. Cancer 17: 583 (1964) and Taswell C, .JImnmunol. 126: 1614 (1981), if single tumorigenic cells were capable offorming tumors, and every transplanted tumorigenic cell gave rise to atumor. Therefore, this calculation may underestimate the frequency ofthe tumorigenic cells (i.e., solid tumor stem cells), since thecalculation does not take into account cell-cell interactions and localenvironment factors that may influence engraftmnent. CD44⁺CD24^(+/low)LINEAGE⁻ populations and CD44⁺CD24^(−/low)LINEAGE⁻ cells were isolatedby flow cytometry as described in FIG. 1.

Limiting dilution analysis of MCF-7 cells showed that the proportion ofclonogenic unsorted cells from this cell line was similar to that ofsorted, enriched breast cancer stem cells from tumors. The mice wereobserved weekly for 4-6½ months or until the mice became sick from thetumors.

Twelve weeks after injection, the injection sites of 20, 000 tumorigenicCD44⁺CD24^(−/low)LINEAGE⁻ cells and 20,000 non-tumorigenicCD44⁺CD24^(+/low)LINEAGE⁻ cells were examined by histology. TheCD44⁺CD24^(−/low)LINEAGE⁻ injection sites contained tumors approximately1 cm in diameter while the CD44⁺CD24⁺LINEAGE⁻ injection sites containedno detectable tumors. Only normal mouse mammary tissue was seen byhistology at the sites of the CD44⁺CD24⁺LINEAGE⁻ injections (FIG. 1 i),whereas the tumors formed by CD44⁺CD24^(−/low)LINEAGE⁻ cells containedmalignant cells as judged by hematoxylin and eosin stained sections(FIG. 1 j). Even when CD44⁺CD24⁺LINEAGE⁻ injection sites fromfifty-eight mice, each administered 1,000-50,000 cells, were examinedafter 16-29 weeks, no tumors were detected. Both the tumorigenic andnon-tumorigenic subsets of LINEAGE⁻ cells from passaged and unpassagedtumors contained >95% cancer cells as judged by Wright staining orPapanicolaou staining and microscopic analysis (FIG. 1 g and FIG. 1 h).

In three of the tumors, further enrichment of tumorigenic activity waspossible by isolating the ESA⁺ subset of the CD44³⁰CD24^(−/low)population. ESA (Epithelial Specific Antigen, Ep-CAM) expressiondistinguishes epithelial cancer cells from benign reactive mesothelialcells. Packeisen J et al., Hybridoma 18: 37-40, 1999). TheCD44⁺CD24^(−/low)LINEAGE⁻ tumorigenic population typically accounted forapproximately 8-25% of viable breast cancer cells, but the data suggestthat in some tumors an even smaller population of tumorigenic cells maybe identified by selecting the ESA subset.

When ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻ cells were isolated from passaged T1,as few as 200 cells consistently formed tumors of approximately 1 cm 6months after injection whereas 2000 ESA⁻CD44⁺CD24 ^(−/low)LINEAGE⁻ cellsor 20,000 CD44⁺CD24⁺ cells always failed to form tumors (TABLE 1). Thesedata suggest that the ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻ population was morethan 50 fold enriched for the ability to form tumors relative tounfractionated tumor cells (TABLE 1). The ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻population accounted for 2-4% of first passage T1cells (2.5-5% of cancercells). The ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻ population (0.6% of cancercells) from unpassaged T5 cells was also enriched for tumorigenicactivity compared to ESA⁻CD44⁺CD24^(−/low)LINEAGE⁻ cells, but both theESA⁺ and ESA⁻ fractions had some tumorigenic activity (TABLE 1). Amongunpassaged T5 cells, as few as 1000 ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻ cellsconsistently formed tumors.

In a comedo subtype breast ductal adenocarcinoma that we designated T7,tumorigenic activity was observed in both the CD44⁺CD24^(−/low) and theCD44⁺CD24⁺ populations (TABLE 1, FIG. 1 f). This suggests that thetumorigenic cells from some patients may differ in cell surface markerexpression.

Phenotypic diversity in tumors arisingfrom solid tumor stem cells. Theability of small numbers of CD44⁺CD24^(−/low)LINEAGE⁻ tumorigenic cellsto give rise to new tumors was reminiscent of the organogenic capacityof normal stem cells. Normal stem cells self-renew and give rise tophenotypically diverse cells with reduced proliferative potential. Totest whether tumorigenic breast cancer cells also exhibit theseproperties, tumors arising from 200 ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻ T1or1,000 CD44^(+CD)24^(−/low)LINEAGE⁻ T2 cells were dissociated andanalyzed by flow cytometry. The heterogeneous expression patterns ofESA, CD44 or CD24 in the secondary tumors resembled the phenotypiccomplexity of the original tumors from which the tumorigenic cells werederived (compare FIG. 2 a and FIG. 2 b with FIG. 2 e and FIG. 2 f).Within these secondary tumors, the CD44⁺CD24^(−/low)LINEAGE⁻ cellsremained tumorigenic, while other populations of LINEAGE⁻ cancer cellsremained non-tumorigenic Passage 2; TABLE 1). Thus tumorigenic cellsgave rise to both additional CD44⁺CD24^(−/low)LINEAGE⁻ tumorigenic cellsas well as to phenotypically diverse non-tumorigenic cells thatrecapitulated the complexity of the primary tumors from which thetumorigenic cells had been derived. These CD44⁺CD24^(−/low)LINEAGE⁻tumorigenic cells from T1, T2 and T3 have now been serially passagedthrough four rounds of tumor formation in mice, yielding similar resultsin each round with no evidence of decreased proliferation. These resultssuggest that CD44⁺CD24^(−/low)LINEAGE⁻ tumorigenic cancer cells undergoprocesses analogous to the self-renewal and differentiation of normalstem cells.

Comparison of the cell cycle status of tumorigenic and non-tumorigeniccancer cells from T1revealed that both exhibited a similar cell cycledistribution (FIG. 2 g and FIG. 2 h). Therefore, neither population wasenriched for cells at a particular stage of the cell-cycle, and thenon-tumorigenic cells were able to undergo at least a limited number ofdivisions in the xenograft model.

The implications of prospectively identifying tumorigenic cancer cells.The tumorigenic CD44⁺CD24^(−/low)LINEAGE⁻ population shares with normalstem cells the ability to proliferate extensively, and to give rise todiverse cell types with reduced developmental or proliferativepotential. The extensive proliferative potential of the tumorigenicpopulation was demonstrated by the ability of as few as 200 passaged or1000 unpassaged ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻ cells to give rise totumors (greater than 1 cm in diameter) that could be seriallytransplanted in NOD/SCID mice. The tumorigenic population from T1, T2and T3 has now been isolated and serially passaged four times throughNOD/SCID mice. This extensive proliferative potential contrasts with thebulk of CD44⁻ and/or CD24⁺cancer cells that lacked the ability to formdetectable tumors. Not only was the CD44⁺CD24^(−/low)LINEAGE⁻ populationof cells able to give rise to additional tumorigenicCD44⁺CD24^(−/low)LINEAGE⁻ cells, but they were also able to give rise tophenotypically diverse non-tumorigenic cells that composed the bulk ofthe tumors. This remained true even after two rounds of serialpassaging. Thus, CD44⁺CD24^(−/low)LINEAGE⁻ cells from most tumors appearto exhibit properties of solid tumor stem cells.

Our data demonstrate there is a hierarchy of solid tumor cells in whichonly a fraction of the cells have the ability to proliferate extensivelywhile other cells have only a limited proliferative potential. Theseresults demonstrate that phenotypically distinct populations of solidtumor cells have an intrinsically greater capacity to proliferateextensively and form new tumors than other populations. In most tumorswe could predict whether cancer cells were tumorigenic or depleted ortumorigenic activity based on marker expression. Although tumorigenicbreast cancer cells were orders of magnitude more likely to form tumorsthan non-tumorigenic breast cancer cells, there may also be a stochasticcomponent to tumorigenicity in the sense that not every injectedtumorigenic cell formed a tumor. Breast cancer cell divisions aregenetically unstable and individual breast cancer cells from thetumorigenic population may sometimes be unable to proliferate as aconsequence of chromosomal aberrations acquired during mitosis. Murphy KL et al., FASEB Journal 14: 2291-2302 (2000).

The observation that eight of nine independent tumors contained a smallpopulation of tumorigenic cells with a common cell surface phenotype hasprofound implications for understanding solid tumor biology and thedevelopment of effective cancer therapies. The inability of currentcancer treatments to cure metastatic disease may be due to ineffectivekilling of tumorigenic cells. If the non-tumorigenic cells arepreferentially killed by particular therapies, then tumors may shrinkbut the remaining tumorigenic cells will drive tumor recurrence. Byfocusing on the tumorigenic population, one can identify criticalproteins that are expressed by virtually all of the tumorigenic cells ina particular tumor. The prospective identification of the tumorigeniccancer cells should allow the identification of more effectivetherapeutic targets, diagnostic markers that detect the dissemination oftumorigenic cells, and more effective prognostic markers, by focusing onthe tumorigenic cells rather than on more functionally heterogeneouscollections of cancer cells.

Notch4 as a therapeutic target. We looked for the expression of proteinsthat may modulate key biological functions of the tumorigenic cells.Activation of the Notch receptor has previously been implicated inbreast cancer and Notch signaling plays a role in transformation ofcells transfected with an activated Ras oncogene. Berry L W et al.,Development 124(4):925-36 (1997); Morrison S J et al., Cell 101(5):499-510 (2000). Given the analogous properties of tumorigenic cancercells and normal stem cells, we focussed on targets such as the Notchsignaling pathway that are known to regulate the self-renewal of avariety of normal stem cells and the proliferation of cancer cell lines.

We have discovered that Notch4 plays a role both in tumorigenesis.Within an individual solid tumor, only a small subpopulation oftumorigenic cells expresses high levels of Notch4. An antibody thatrecognizes Notch4 blocks the growth of breast cancer tumor cells invitro and in vivo. In one embodiment, the antibody binds to theextracellular domain of Notch4. In a particular embodiment, the antibodybinds to the polypeptide region LLCVSVVRPRGLLCGSFPE(LeuLeuCysValSerValValArgProArgGlyLeuLeuCysGlySerPheProGlu) (SEQ IDNO:1). However, any anti-Notch4 antibody that inhibits Notch activationcan be used to impair tumor survival.

We found by RT-PCR that T1 CD44⁺CD24^(−/low)LINEAGE⁻ tumorigenic cellsexpressed Notch4 (FIG. 3 a). We therefore tested the effect of Notchactivation in breast cancer cells by exposing the cells in culture to asoluble form of the Notch ligand Delta, Delta-Fc. Morrison S J et al.,Cell 101: 499-510 (2000). We found that soluble Delta increased thenumber of colonies formed by unfractionated T1cancer cells in culturefive-fold (FIG. 3 b). Thus, Notch activation promoted the survival orproliferation of clonogenic cancer cells, i.e., solid tumor stem cells.

To test whether inhibition of Notch4 signaling would reduce survival orproliferation, we exposed the cells to a polyclonal, blocking antibodyagainst Notch4 that reduced Notch pathway reporter gene activation (FIG.3 c). The anti-Notch4 antibody which was purchased from Santa CruzProducts (Santa Cruz, Calif. USA). The antibody binds to the polypeptideregion LLCVSVVRPRGLLCGSFPE(LeuLeuCysValSerValValArgProArgGlyLeuLeuCysGlySerPheProGlu) (SEQ IDNO:1). For the Notch reporter assay, the HES-1—Luciferase reporterconstruct was made as described by Liu A Y et al., Proc. Natl. Acad.Sci. USA 94: 10705-10710 (1997). The fragment of the HES-1 murine genebetween −194 and +160 was amplified by PCR and subdloned into a pGL2basic vector (Promega) between the KpnI and Bgl II sites. MCF-7 cellswere co-transfected with the HES-1-luc construct and pSV2Neo andselected in medium containing geneticin.

For RT-PCR, RNA was isolated using Trizol (Gibco BRL). For the Notch4gene expression analysis, reverse transcription of 0.2 mg RNA isolatedfrom T1, MCF-7 and MCF-10A cells , was done using a gene specific anchorprimer 5′-TCCTCCTGCTCCTACTCCCGAGA-3′ (SEQ ID NO: 2). The Notch4 fragmentwas amplified using the following primers:5′-TGAGCCCTGGGAACCCTCGCTGGATGGA-3′ (SEQ ID NO: 3) and5′-AGCCCCTTCCAGCAGCGTCAGCAGAT-3′ (SEQ ID NO: 4).

The transfected MCF-7 cells were cocultivated in 12-well plates in thepresence and absence of the Notch4 polyclonal antibody (Santa Cruz; 20μg/ml final concentration), soluble Delta-Fc (Morrison S J et al., Cell101: 499-510 (2000)) or the Notch4 antibody blocking peptide (4 mg/100ml final concentration, Santa Cruz Products), LLCVSVVRPRGLLCGSFPE(LeuLeuCysValSerValValArgProArgGlyLeuLeuCysGlySerPheProGlu) (SEQ IDNO:1).

Luciferase assays were performed as described by Jarriault S et al.,Nature 377: 355-358 (1995). Delta-Fc or Fc control proteins wereconcentrated from the supernatant of 293 cells that were engineered tosecrete them according to the methods of Morrison S J et al., Cell 101:499-510 (2000). Delta-Fc or Fc control proteins were added to breastcancer cell cultures along with a cross-linking anti-Fc antibody(Jackson Imunoresearch) as previously described by Morrison S J et al.,Cell 101: 499-510 (2000).

When cells were exposed to this anti-Notch4 antibody, colony formationwas almost completely inhibited (FIG. 3 b). This inhibition was nearlycompletely eliminated by pre-incubation of the antibody with the Notch4peptide against which the antibody was generated, which presumablyprevented binding of the antibody to Notch4 on the cell surface (FIG. 3b). The anti-Notch4 antibody also inhibited colony formation by theMCF-7 breast cancer cell line, but not the MCF-10 cell line (Soule H Det al., Cancer Research 50, 6075-6086 (1990)) that was derived fromnormal breast epithelium. To determine whether the anti-Notch4 antibodyinhibited tumor formation, 100-500 ESA³⁰ CD44⁺CD24^(−/low)LINEAGE⁻ cellsincubated with either control buffer or the anti-Notch4 antibody wereinjected into mice. nine of eleven injections of 200-500 untreated cellsand one of eleven injections of 100 untreated cells formed tumors (FIG.3 d). By contrast, injection of 100 or 200 cells treated withanti-Notch4 antibody failed to form tumors and tumor formation by 500antibody-treated cells was delayed relative to control cells (FIG. 3 d).

Notch4 signaling provides a survival signal to tumor-initiating cells.We next studied the mechanism by which anti-Notch4 antibody inhibitedcolony formation. Notch stimulation has been shown to promoteself-renewal in some circumstances, inhibit proliferation in othercircumstances, and to promote survival in other cases. To distinguishbetween these possibilities, unfractionated cancer cells isolated fromfour tumors, MCF-7 cells and MCF-10 cells were analyzed forproliferation and cell death after exposure to the anti-Notch4 antibody.There was no significant difference in the cell cycle distribution ofMCF-7 cells (which expressed Notch4, supplemental FIG. 1), exposed tothe anti-Notch4 antibody when compared to untreated cells twenty-fourhours after antibody exposure (FIG. 4 a). However, exposure of MCF-7cells, unfractionated tumor cells isolated from T10, or T1ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻ breast cancer tumorigenic cells, but notMCF10 cells or unfractionated tumor cells isolated from T7 and T8, tothe anti-Notch4 blocking antibody led to the accumulation of cells withdegraded DNA characteristic of apoptosis and to the activation ofcaspase {fraction (3/7)} in a significant percentage of the cellsthirty-six hours after antibody exposure (FIG. 4 b and FIG. 4 c).

For the apoptosis assays, tumorigenic T1 cells(ESA⁺CD44⁺CD24^(−/low)LINEAGE⁻) or LINEAGE⁻ tumor cells from T7, T8 andT10 were sorted by flow cytometry and grown on collagen coated tissueculture plates. The T10 tumorigenic cells have not yet beencharacterized. Anti-Notch4 polyclonal antibody (Santa Cruz , Calif. USA)was then added to the medium (20 mg/ml final concentration) while PBSwas added to the control plates. To block the anti-Notch4 antibody, theanti-Notch4 antibody was pre-incubated with the blocking peptide (SantaCruz, Calif. USA) on ice for 30 minutes after which it was added to themedium. After 48 hrs, cells were trypsinized and collected. 10⁵ cellswere suspended in HBSS 2% heat inactivated calf serum and then assayedfor apoptosis using FAM-DEVD-FMK, a caroxyfluorescein labeled peptidesubstrate specific to caspases 3 and 7 (CaspaTag™ Caspase Activity Kit,Intergen Company, New York USA) to detect active caspases in livingcells. Caspase positive cells were distinguished from the negative onesusing FACSVantage flow cytometer (Becton Dickinson, California USA). PIstaining for cell cycle and apoptosis was performed as described byClarke M F et al., Proc. Natl. Acad. Sci. USA 92:11024-11028, (1995).

These data suggest that, in some de novo human tumors, Notch pathwayactivation provides a necessary survival signal to the tumorigenicpopulation of breast cancer cells.

Maniac Fringe as a therapeutic target for breast cancer stem cells.Proteins with knife-edge names such as Jagged (Shimizu et al., Journalof Biological Chemistry 274(46) 32961-9 (1999); Jarriault et al.,Molecular and Cellular Biology 18: 7423-7431 (1998)), Serrate, and Delta(and variants of each, such as Delta1, Delta2, Delta3, Delta4, Jagged 1and Jagged2, LAG-2 and APX-1 in C. elegans), bind to the Notch receptorand activate a downstream signaling pathway that prevents neighboringcells from becoming neural progenitors. The recently identified ligandis D114 is a Notch ligand of the Delta family expressed in arterialendothelium. Shutter et al., Genes Dev 14(11): 1313-8 (2000)).

Notch ligands may bind and activate Notch family receptorspromiscuously. The expression of other genes, like Fringe family members(Panin et al, Nature 387(6636): 908-912 (1997)), may modify theinteractions of Notch receptors with Notch ligands. Numb family membersmay also modify Notch signaling intracellularly.

Ligand binding to Notch results in activation of apresenilin-1-dependent gamma-secretase-like protein that cleaves Notch.De Strooper et al., Nature 398: 518-522 (1999), Mumm et al., MolecularCell. 5: 197-206 (2000). Cleavage in the extracellular region mayinvolve a furin-like convertase. Logeat et al., Proceedings of theNational Academy of Sciences of the USA 95: 8108-8112 (1998). Theintracellular domain is released and transactivates genes by associatingwith the DNA binding protein RBP-J. Kato et al., Development 124:4133-4141 (1997)). Notch1, Notch2 and Notch4 are thought totransactivate genes such as members of the Enhancer of Split (HES)family, while Notch3 signaling may be inhibitory. Beatus et al.,Development 126: 3925-3935 (1999). Finally, secreted proteins in theFringe family bind to the Notch receptors and modify their function.Zhang & Gridley, Nature 394 (1998).

Inhibitors of Notch signaling (such as Numb and Numb-like; or antibodiesor small molecules that block Notch activation) can be used in themethods of the invention to inhibit solid tumor stem cells. In thismanner, the Notch pathway is modified to kill or inhibit theproliferation of solid tumor stem cells.

Since the Notch signaling pathway appears to play a critical role in theproliferation of T1 cancer stem cells and MCF-7 cells, we determined theexpression of Notch4 and members of the Fringe family by differentpopulations of Tumor 1 cancer cells. Flow cytometry showed that both thetumorigenic and non-tumorigenic cancer cells expressed Notch4. We nextexamined two tumors for expression of members of the Fringe family. Thethree Fringe proteins, Manic, Lunatic and Radical, all glycosylate Notchreceptors and modulate receptor signaling. However, the effect of aparticular Fringe on signal transduction via each of the four Notchreceptors can differ. Furthermore, each Fringe is thought to glycosylatea particular Notch receptor at different sites, resulting in adifferential response to a particular ligand.

RNA was isolated from solid tumor cells using Trizol (Gibco BRL,Rockfill, Md.). After reverse transcription, the EGF-R and the Her2/neufragments were amplified using the following primers: EGF-R,5′-GCCAGGAATTGAGAGTCTCA-3′ (SEQ ID NO:5), 5′-AAGCCTGTTATTCTGCCTTTTA-3′(SEQ ID NO:6), 5′-CCACCAATCCAACATCCAGA-3′ (SEQ ID NO:7) and5′-AACGCCTGTCATAGAGTAG-3′ (SEQ ID NO:8); Her2/neu,5′-CACAGGTTACCTATACATCT-3′ (SEQ ID NO:9), 5′-GGACAGCCTGCCTGACCTCA-3′(SEQ ID NO:10), 5′-CCACAGGGAGTATGTGAATG-3′ (SEQ ID NO:11), and5′-TTTGCCGTGGGACCCTGAGT-3′ (SEQ ID NO:12) respectively. The RT-PCR forthe Fringe transcripts were done using the following external primers,for Manic fringe, 5′-GGCTGAATTGAAAAAGGGCAG-3′ (SEQ ID NO:13) and5′-AGCAGGAAGATGGGGAGTGG-3′ (SEQ ID NO:14), for Radical Fringe,5′-CCGAGAGGGTCCAGGGTGGC-3′ (SEQ ID NO:15)and 5′-CCTGAGGGAGCCCACTGAGC-3′(SEQ ID NO:16), and for Lunatic Fringe 5′-CCAGCCTGGACAGGCCCATC-3′ (SEQID NO:17), and 5′-ACGGCCTGCCTGGCTTGGAG-3′ (SEQ ID NO:18) respectivelyand the following internal primers.

RT-PCR using 0.1 ug of unseparated tumor RNA demonstrated that T1 cellsexpressed Manic Fringe, Radical Fringe and Lunatic Fringe whereas RT-PCRof 100 ESA⁺B38.1⁺CD24^(−/lo)LINEAGE⁻ (tumorigenic) cells demonstratedthat these cells expressed Manic Fringe, but not Lunatic Fringe orRadical Fringe. When examined by single cell RT-PCR, all sixT1tumorigenic cells expressed Manic Fringe, but only two of sixnon-tumorigenic cells did so. By contrast, all of the non-tumorigenic,but none of the tumorigenic, single cells examined expressed LunaticFringe and Radical Fringe. Fringe expression by unpassaged T5 stem cellsand non-tumorigenic cells was determined to see if there was adifference in expression by the different populations of unpassagedbreast cancer cells. Single cell RT-PCR showed that all six of the T5breast cancer stem cells tested expressed Manic Fringe, but only ⅙ ofthe cells expressed Lunatic Fringe and only one of six cells testedexpressed Radical Fringe respectively. By contrast, all of thenon-tumorigenic cells tested expressed Lunatic Fringe and five of sixexpressed Radical Fringe, but only one of six cells expressed ManicFringe. Thus, the expression of the different Fringe genes by thetumorigenic and non-tumorigenic unpassaged T5 cells reflected thepattern seen in the passaged T1 cells. Manic Fringe has been implicatedin oncogenic transformation. These data demonstrate the differentialexpression by tumorigenic and non-tumorigenic neoplastic cells of genesinvolved in a biologically relevant pathway that appears to regulatetumorigenesis in these cells. Whether the different Fringe genes play adirect role in breast cancer cell fate decisions or their differentialexpression is simply associated with a particular cell populationremains to be tested.

Selective targeting of solid tumor stein cells. We determined theexpression of EGF-R, Her2/neu, Notch4, Manic Fringe, Lunatic Fringe andRadical Fringe by tumorigenic breast cancer cells (i.e., solid tumorstem cells, in particular Tumor 1 (T1) cells) EGF-R and HER2/neu arepotential therapeutic targets that have been implicated in breast cancercell proliferation.

Flow cytometry was used to isolate subpopulations of T1cells that hadbeen passaged once in NOD/SCID mice. Cells were stained with anti-EGF-R,anti-CD24, anti-Lineage, anti-mouse-H2K, and 7AAD or anti-HER2/neu,anti-CD24, anti-Lineage, anti-mouse-H2K, and 7AAD. Dead cells (7AAD⁺),mouse cells (H2K⁺) and LINEAGE⁺ cells were eliminated from all analyses.RT-PCR using nested primers was also performed to detect EGF-R or todetect HER2/neu in one cell per sample in panels or ten cells per samplein panels.

By focusing on the tumorigenic population of cells in T1, we were ableto identify Her2/neu Notch4 and Manic Fringe, while potentiallyeliminating EGF-R Radical Fringe and Lunatic Fringe, as possibletherapeutic targets in this particular tumor. While most of thetumorigenic cells expressed detectable levels of HER2/neu protein andmRNA, we were not able to detect expression of EGF-R in most tumorigeniccells.

Tumorigenic T1 cells stained with lower levels of anti-EGF-R antibodythan non-tumorigenic cells, and EGF-R expression could not be detectedat the single cell level in tumorigenic cells. To test whether cellsthat did not express detectable levels of the EGF-R were tumorigenic,1,000-2,000 tumorigenic cells were also sorted with respect to EGFRexpression and injected into NOD/SCID mice. Tumors formed in four out offour cases, confirming that the EGF-R⁻ cells are tumorigenic. Incontrast, we could not detect a substantial difference in HER2/neuexpression between tumorigenic and non-tumorigenic T1 cells. Asexpected, 1,000-2,000 HER2/neu⁺ cells gave rise to tumors in four out offour cases. These observations suggest that there can be differences inthe expression of therapeutic targets between the tumorigenic andnon-tumorigenic populations.

Since therapies that kill only non-tumorigenic cancer cells may producetemporary tumor regression but will not be able to eradicate the tumor,these results suggest that agents that kill HER2/neu expressing cellsmight be more effective than those that kill EGF-R expressing cells inthis tumor. Other breast cancer tumors may manifest different patternsof expression of these genes. Thus, theprospective identification andisolation of tumorigenic cells should allow a more focused biological,biochemical and molecular characterization of the factors critical fortumor formation and permit the specific targeting of therapeutic agentsto this cell population, resulting in the development of more effectivecancer treatments.

Solid stem cells and solid stem cell progeny of the invention can beused in methods of determining the effect of a biological agents onsolid tumor cells, e.g., for diagnosis, treatment or a combination ofdiagnosis and treatment. The term “agent” or “compound” refers to anyagent (including a virus, protein, peptide, amino acid, lipid,carbohydrate, nucleic acid, nucleotide, drug, antibody, prodrug, other“biomolecule” or other substance) that may have an effect on tumor cellswhether such effect is harmful, beneficial, or otherwise. The ability ofvarious biological agents to increase, decrease, or modify in some otherway the number and nature of the solid tumor stem cells and solid tumorstem cell progeny can be assayed by methods known to those of skill inthe drug discovery art.

In one embodiment, a pharmaceutical composition containing a Notch4ligand, an anti-Notch4 antibody, or other therapeutic agent that acts asan agonist or antagonist of proteins in the Notch signaltransduction/response pathway can be administered by any effectivemethod. For example, a physiologically appropriate solution containingan effective concentration of anti-Notch therapeutic agent can beadministered topically, intraocularly, parenterally, orally,intranasally, intravenously, intramuscularly, subcutaneously or by anyother effective means. In particular, the anti-Notch therapeutic agentmay be directly injected into a target cancer or tumor tissue by aneedle in amounts effective to treat the tumor cells of the targettissue. Alternatively, a solid tumor present in a body cavity such as inthe eye, gastrointestinal tract, genitourinary tract (e.g., the urinarybladder), pulmonary and bronchial system and the like can receive aphysiologically appropriate composition (e.g., a solution such as asaline or phosphate buffer, a suspension, or an emulsion, which issterile) containing an effective concentration of anti-Notch4therapeutic agent via direct injection with a needle or via a catheteror other delivery tube placed into the cancer or tumor afflicted holloworgan. Any effective imaging device such as X-ray, sonogram, orfiber-optic visualization system may be used to locate the target tissueand guide the needle or catheter tube. In another alternative, aphysiologically appropriate solution containing an effectiveconcentration of anti-Notch therapeutic agent can be administeredsystemically into the blood circulation to treat a cancer or tumor thatcannot be directly reached or anatomically isolated. All suchmanipulations have in common the goal of placing the anti-Notch4 agentin sufficient contact with the target tumor to permit the anti-Notch4agent to contact, transduce or transfect the tumor cells (depending onthe nature of the agent).

In treating a cancer patient who has a solid tumor, a therapeuticallyeffective amount of an anti-Notch therapeutic agent can be administered.A “therapeutically effective” dose refers to that amount of the compoundsufficient to result in amelioration of symptoms or a prolongation ofsurvival in a patient. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit largetherapeutic indices are preferred. The data obtained from these cellculture assays and animal studies can be used in formulating a range ofdosage for use in humans. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography (HPLC).

In another embodiment, a biomolecule or biological agent selectivelytargeted to a solid tumor stem cell can use gene therapy strategies. Forexample, the biomolecule can be a gene therapy suicide vector targetedto solid tumor stem cells using markers expressed by the solid tumorstem cells. In one embodiment, the vector is an adenoviral vector whichhas been redirected to bind to the B38.1 marker. We conjugatedanti-fiber and the B38.1 antibodies with the Prolinx (Prolinx, Inc.,Bothell, Wash., USA) method (see Douglas J T et al., NatureBiotechnology. 14(11):1574-8 (1996)). When we mixed the modifiedanti-knob and anti-B38.1 antibodies together, they became cross-linkedand generated the bi-specific conjugate. The anti-fiber antibody part ofthe conjugate can bind to the adenovirus, while the anti-B38.1 moietycan bind to the breast cancer stem cell. Incubation of the AdLacZ viruswith the anti-fiber alone blocks the infectivity of the virus. Theinfectivity of virus incubated with the bi-specific conjugate isrestored only in the cells that express high levels of the B38.1antigen. The re-targeting is specific, because it can be inhibited byfree B38.1 antibody. The conclusion is that a bi-specific conjugate canmodifies the infectivity of a vector, blocking its natural tropism anddirecting the infection to cells that express the solid tumor stem cellsurface marker.

One skilled in the oncological art of can understand that the vector isto be administered in a composition comprising the vector together witha carrier or vehicle suitable for maintaining the transduction ortransfection efficiency of the chosen vector and promoting a safeinfusion. Such a carrier may be a pH balanced physiological buffer, suchas a phosphate, citrate or bicarbonate buffers a saline solution, a slowrelease composition and any other substance useful for safely andeffectively placing the targeted agent in contact with solid tumor stemcells to be treated.

Depending on the specific conditions being treated, agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 20th ed. (Mack Publishing Co., Easton, Pa.).Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

For injection, the agents of the invention maybe formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, capsules, or solutions. The pharmaceutical compositions of thepresent invention may be manufactured in a manner that is itself known,e.g., by means of conventional mixing, dissolving, granulating,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition (see e.g.Fingl et al., In The Pharmacological Basis of Therapeutics, Ch. 1, pg. 1(1975)). The attending physician would know how to and when toterminate, interrupt, or adjust administration due to toxicity, or toorgan dysfunctions. Conversely, the attending physician would also knowto adjust treatment to higher levels if the clinical response were notadequate (precluding toxicity). The magnitude of an administrated dosein the management of the clinical disorder of interest can vary with theseverity of the condition to be treated and the route of administration.See, Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals,12 Edition (CRC Press 1996); Physicians'Desk Reference 55th Edition(2000)). The severity of the condition may, for example, be evaluated,in part, by appropriate prognostic evaluation methods. Further, the doseand perhaps dose frequency, also vary according to the age, body weight,and response of the individual patient. A program comparable to thatdiscussed above may be used in veterinary medicine.

The details of one or more embodiments of the invention have been setforth in the accompanying description above. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Other features, objects, and advantages ofthe invention will be apparent from the description and from the claims.

In the specification and the appended claims, the singular forms includeplural referents. Unless defined otherwise in this specification, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. All patents and publications cited in thisspecification are incorporated by reference.

1. A method for reducing the size of a solid tumor, comprising the stepof: contacting the cells of the solid tumor with a therapeuticallyeffective amount of an agent directed against a Notch4 polypeptide. 2.The method of claim 1, wherein the therapeutically effective amount isan amount sufficient to cause cell death of or inhibit the proliferationof solid tumor stem cells in the solid tumor.
 3. The method of claim 1,wherein the agent is an antibody, peptide or small molecule directedagainst a Notch4 polypeptide.
 4. The method of claim 3, wherein theantibody, peptide or small molecule is directed against theextracellular domain of Notch4.
 5. A method for reducing the size of asolid tumor, comprising: contacting the cells of the solid tumor with atherapeutically effective amount of an agent that modulates the activityof a Notch4 ligand.
 6. The method of claim 5, wherein the Notch4 ligandis selected from the group consisting of Delta 1, Delta 2, Delta-likeligand 4 (D114), Jagged 1 and Jagged
 2. 7. The method of claim 5,wherein the agent is a Notch ligand agonist.
 8. The method of claim 5,wherein the agent is a Notch ligand antagonist.
 9. A method for reducingthe size of a solid tumor, comprising: contacting the cells of the solidtumor with a therapeutically effective amount of an agent that modulatesthe activity of Maniac Fringe.
 10. The method of claim 9, wherein theagent is a Maniac Fringe agonist.
 11. The method of claim 9, wherein theagent is a Maniac Fringe antagonist.
 12. A method for killing orinhibiting the proliferation of solid tumor stem cells, comprising thestep of: contacting the cells of a solid tumor with an agent orcombination of agents selectively targeted to the solid tumor stem cellsof the solid tumor, wherein the agent or combination of agents kills orinhibits the proliferation of solid tumor stem cells.
 13. The method ofclaim 12, further comprising the step of: identifying the death of orthe prevention of the growth of solid tumor stem cells in the solidtumor following contact by the agent or combination of agents.
 14. Themethod of claim 12, wherein the killing is by the activation of celldeath in the solid tumor stem cells.
 15. The method of claim 14, whereinthe cell death is apoptosis.
 16. The method of claim 12, wherein theagent or combination of agents inhibits Notch4 signaling.
 17. The methodof claim 12, wherein the agent is an antibody, peptide or small moleculedirected against a Notch4 polypeptide.
 18. The method of claim 12,wherein the antibody, peptide or small molecule is directed against theextracellular domain of Notch4.
 19. The method of claim 12, wherein theagent or combination of agents modulates the activity of a Notch4ligand.
 20. The method of claim 19, wherein the Notch4 ligand isselected from the group consisting of Delta 1, Delta 2, Delta-likeligand 4 (D114), Jagged 1 and Jagged
 2. 21. The method of claim 12,wherein the agent or combination of agents modulates the activity ofManiac Fringe.
 22. The method of claim 12, wherein the solid tumor stemcells express at least one marker selected from the group consisting ofCD44, epithelial specific antigen (ESA), and B38.1.
 23. The method ofclaim 12, wherein the solid tumor stem cells express the cell surfacemarker CD44.
 24. The method of claim 12, wherein the solid tumor stemcells express the cell surface marker epithelial specific antigen (ESA).25. The method of claim 12, wherein the solid tumor stem cells expressthe cell surface marker B38.1.
 26. The method of claim 12, wherein thesolid tumor stem cells express lower levels of the marker CD24 than themean expression of CD24 by non-tumorigenic cancer cells of the solidtumor.
 27. The method of claim 12, wherein the solid tumor stem cellsfail to express at least one LINEAGE marker selected from the groupconsisting of CD2, CD3, CD10, CD14, CD16, CD31, CD45, CD64, and CD140b.28. The method of claim 12, wherein the solid tumor is an epithelialcancer or a sarcoma
 29. The method of claim 28, wherein the epithelialcancer is a breast cancer or an ovarian cancer.
 30. A method forreducing the size of a solid tumor, comprising the step of: contactingthe cells of the solid tumor in vivo with an agent or combination ofagents selectively targeted to the solid tumor stem cells of the solidtumor, wherein the agent or combination of agents kills or inhibits theproliferation of solid tumor stem cells.
 31. The method of claim 30,further comprising the step of: identifying the death of or theprevention of the growth of solid tumor stem cells in the solid tumorfollowing contact by the agent or combination of agents.
 32. The methodof claim 30, wherein the killing is by the activation of cell death inthe solid tumor stem cells.
 33. The method of claim 32, wherein the celldeath is apoptosis.
 34. The method of claim 30, wherein the agent orcombination of agents inhibits Notch-4 signaling.
 35. The method ofclaim 30, wherein the agent is an antibody, peptide or small moleculedirected against a Notch4 polypeptide.
 36. The method of claim 35,wherein the antibody, peptide or small molecule is directed against theextracellular domain of Notch4.
 37. The method of claim 30, wherein theagent or combination of agents modulates the activity of a Notch ligand.38. The method of claim 30, wherein the Notch4 ligand is selected fromthe group consisting of Delta 1, Delta 2, Delta-like ligand 4 (D114),Jagged 1 and Jagged
 2. 39. The method of claim 30, wherein the agent orcombination of agents modulates the activity of Maniac Fringe.
 40. Themethod of claim 30, wherein the solid tumor stem cells express at leastone marker selected from the group consisting of CD44, epithelialspecific antigen (ESA). and B38.1.
 41. The method of claim 30, whereinthe solid tumor stem cells express the cell surface marker CD44.
 42. Themethod of claim 30, wherein the solid tumor stem cells express the cellsurface marker epithelial specific antigen (ESA).
 43. The method ofclaim 30, wherein the solid tumor stem cells express the cell surfacemarker B38.1.
 44. The method of claim 30, wherein the solid tumor stemcells fail to express at least one LINEAGE marker selected from thegroup consisting of CD2, CD3, CD10, CD14, CD16, CD31, CD45, CD64, andCD140b.
 45. The method of claim 30, wherein the solid tumor stem cellsexpress lower levels of the marker CD24 than the mean expression of CD24by non-tumorigenic cancer cells of the solid tumor.
 46. The method ofclaim 30, wherein the solid tumor is an epithelial cancer or a sarcoma.47. The method of claim 46, wherein the epithelial cancer is a breastcancer or an ovarian cancer.
 48. A method for selectively targeting asolid tumor stem cell, comprising the steps of: (a) identifying a markerpresent on a solid tumor stem cell; (b) obtaining a biomolecule or setof biomolecules that selectively binds to the marker present on thesolid tumor stem cell.
 49. The method of claim 48, wherein thebiomolecule genetically modifies the targeted solid tumor stem cell. 50.The method of claim 49, wherein the genetic modification results insolid tumor stem cell death.
 51. The method of claim 48 wherein thebiomolecule or set of biomolecules comprises a bi-specific conjugate.52. The method of claim 48, wherein the biomolecule or set ofbiomolecules comprises an adenoviral vector.
 53. The method of claim 49,wherein the adenoviral vector is selectively targeted to a solid tumorstem cell.
 54. A biomolecule or set of biomolecules that is selectivelytargeted to solid tumor stem cell.
 55. The method of claim 54, whereinthe biomolecule genetically modifies the targeted solid tumor stem cell.56. The method of claim 55, wherein the genetic modification results insolid tumor stem cell death.
 57. The method of claim 54, wherein thebiomolecule or set of biomolecules comprises a bi-specific conjugate.58. The method of claim 54, wherein the biomolecule or set ofbiomolecules comprises an adenoviral vector.
 59. The method of claim 58,wherein the adenoviral vector is selectively targeted to a solid tumorstem cell.
 60. A method for forming a tumor in an animal, comprising:introducing a cell dose of purified solid tumor stem cells into theanimal, wherein: (a) the solid tumor stem cell is derived from a solidtumor; (b) the solid tumor stem cell population is enriched at least2-fold relative to unfractionated tumor cells.
 61. The method of claim60, wherein the animal is an immunocompromised animal.
 62. The method ofclaim 60, wherein the animal is a mammal.
 63. The method of claim 62,wherein the mammal is an immunocompromised mammal.
 64. The method ofclaim 62, wherein the mammal is a mouse.
 65. The method of claim 64,wherein the mouse is an immunocompromised mouse.
 66. The method of claim65, wherein the immunocompromised mouse is selected from the groupconsisting of nude mouse, SCID mouse, NOD/SCID mouse, Beige/SCID mouse;and β2 microglobin deficient NOD/SCID mouse.
 67. The method of claim 60,wherein the number of cells in the cell dose is between about 100 cellsand about 5×10⁵ cells.
 68. The method of claim 60, wherein the number ofcells in the cell dose is about between about 100 cells and 500 cells.69. The method of claim 60, wherein the number of cells in the cell doseis between about 100 cells and 200 cells.
 70. The method of claim 60,wherein the number of cells in the cell dose is about 100 cells.
 71. Themethod of claim 60, wherein the solid tumor stem cell expresses at leastone marker selected from the group consisting of CD44, epithelialspecific antigen (ESA), and B38.1.
 72. The method of claim 60, whereinthe solid tumor stem cell expresses the cell surface marker CD44. 73.The method of claim 60, wherein the solid tumor stem cell expresses thecell surface marker epithelial specific antigen (ESA).
 74. The method ofclaim 60, wherein the solid tumor stem cell expresses the cell surfacemarker B38.1.
 75. The method of claim 60, wherein the solid tumor stemcell expresses lower levels of the marker CD24 than the mean expressionof CD24 by non-tumorigenic cancer cells derived from the solid tumor.76. The method of claim 60, wherein the solid tumor stem cell does notexpress detectable levels of one or more LINEAGE markers, wherein aLINEAGE marker is selected from the group consisting of CD2, CD3, CD10,CD14, CD16, CD31, CD45, CD64, and CD140b.